Myocardial ischemia is characterized by decreased oxidative metabolism and accumulation of long chain acyl-CoA and acylcarnitines. Subsarcolemmal and myofibrillar mitochondrial isolation will be carried out to determine possible differential effects of ischemia on these populations. Effects of energy charge (ATP/ADP) on oxidative metabolism of Krebs cycle substrates and long chain fatty acids, and changes in apparent affinity of mitochondria for ADP will be correlated with the kinetics of the carnitine acyltransferase reaction. To determine if accumulation of acyl-CoA, acylcarnitine and increased intramitochondrial NADH/NAD ion produce mitochondrial alterations carried through isolation, control experiments will include preincubation with varying concentrations of acyl-CoA/acylcarnitine in the presence and absence of glutamate-malate or reconstituted malate-aspartate shuttle. Nonspecific swelling and lytic effects will be assessed as well as reversible association of lipid with mitochondria membranes via inclusion of BSA in the incubation and wash. The effects of incubation on the carnitine acyltransferase reactions as well as phosphorylating respiration (at limiting ADP) will be determined. Since increased association of acyl-CoA with mitochondria may act to competitively inhibit both adenine nucleotide transport and carnitine acyltransferase, the reactivity of the external and inner membrane transferase will be assessed following ischemia and under conditions that ischemia. The sensitivity of heart mitochondrial beta-oxidation to limiting NAD ion will be measured at varying energy charge and NADH/NAD ratios generated by the reconstituted malate aspartate shuttle. Suppression of beta-oxidation (measured by incorporation of C14 label into CO2 and acid soluble products) will be correlated with changes in the apparent affinity of mitochondria for ADP during phosphorylating respiration.